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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-03-27 to 2017-04-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Qualifier:
according to guideline
Guideline:
other: Direct Peptide Reactivity Assay (DPRA) for Skin Sensitization Testing, DB-ALM Protocol n°154, January 12, 2013
GLP compliance:
yes (incl. QA statement)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
Type of study:
other: (in chemico) reactivity against synthetic peptides with a thiol or amino group

Test material

Constituent 1
Chemical structure
Reference substance name:
N-acetylhexanelactam
EC Number:
217-565-6
EC Name:
N-acetylhexanelactam
Cas Number:
1888-91-1
Molecular formula:
C8H13NO2
IUPAC Name:
N-acetylhexanelactam
Test material form:
liquid
Details on test material:
Batch: not yet known
Specific details on test material used for the study:
Name: N-acetylhexanelactam
Batch No.: ESD0024613
Chemical name: 1-acetylazepan-2-one
CAS No 1888-91-1
Purity 98.1% (GC)
Physical State: liquid
Molecular Weight: 155.2 g/mol
Colour: light yellow clear
Storage Conditions: room temperature
Stability: stable at storage conditions
Expiry Date: 06 May 2018
Safety Precautions: The routine hygienic procedures are sufficient to assure personnel health and safety.

In chemico test system

Details on the study design:
The in chemico direct peptide reactivity assay (DPRA) enables detection of the sensitising potential of a test item by quantifying the reactivity of test chemicals towards synthetic peptides containing either lysine or cysteine.

Results and discussion

Positive control results:
The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean peptide depletion of the positive control for the cysteine peptide was between 60.8% and 100% (74.90 %). The mean peptide depletion of the positive control for the lysine peptide was between 40.2% and 69.0% (54.93%).

In vitro / in chemico

Resultsopen allclose all
Key result
Run / experiment:
other: cysteine run
Parameter:
other: mean peptide depletion [%] cysteine run
Value:
3.82
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: lysine run
Parameter:
other: mean peptide depletion [%] lysine run
Value:
19.69
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
Acceptance Criteria

The run meets the acceptance criteria if:
- the standard calibration curve has a r² > 0.99,
- the mean percent peptide depletion (PPD) value of the three replicates for the positive control is
between 60.8% and 100% for the cysteine peptide and the maximum standard deviation (SD) for the
positive control replicates is < 14.9%,
- the mean percent peptide depletion (PPD) value of the three replicates for the positive control is
between 40.2% and 69.0% for the lysine peptide and the maximum SD for the positive control
replicates is < 11.6%,
- the mean peptide concentration of the three reference controls A replicates is 0.50 ± 0.05 mM,
- the coefficient of variation (CV) of peptide peak areas for the six reference control B replicates and three reference control C replicates in acetonitrile is < 15.0%.

The results of the test item meet the acceptance criteria if:
- the maximum standard deviation (SD) for the test chemical replicates is < 14.9% for the cysteine
percent depletion (PPD),
- the maximum standard deviation (SD) for the test chemical replicates is < 11.6% for the lysine
percent depletion (PPD),
- the mean peptide concentration of the three reference controls C replicates in the appropriate solvent is 0.50 ± 0.05 mM.

Both peptide runs and the test item results met the acceptance criteria of the test.

Any other information on results incl. tables

Cysteine and Lysine Values of the Calibration Curve

Sample

Cysteine Peptide

Lysine Peptide

Peak Area
at 220 nm

Peptide Concentration [mM]

Peak Area
at 220 nm

Peptide Concentration [mM]

STD7

0.0000

0.0000

0.0000

0.0000

STD6

153.8068

0.0167

139.6547

0.0167

STD5

321.6256

0.0334

281.4845

0.0334

STD4

642.2243

0.0667

555.3300

0.0667

STD3

1318.5444

0.1335

1100.1805

0.1335

STD2

2593.4377

0.2670

2176.1533

0.2670

STD1

5075.1016

0.5340

4280.0898

0.5340

 

Depletion of the Cysteine Peptide

Cysteine Peptide

Sample

Peak Area at 220 nm

Peptide Concentration [mM]

Peptide Depletion [%]

Mean Peptide Depletion [%]

SD of Peptide Depletion [%]

CV of Peptide Depletion [%]

Positive Control

1193.1283

0.1239

74.99

74.90

0.25

0.34

1187.7434

0.1233

75.10

1210.7732

0.1258

74.62

Test Item

4595.2026

0.4810

3.67

3.82

0.21

5.37

4592.3970

0.4807

3.73

4577.0308

0.4791

4.05

Depletion of the Lysine Peptide

Lysine Peptide

Sample

Peak Area at 220 nm

Peptide Concentration [mM]

Peptide Depletion [%]

Mean Peptide Depletion [%]

SD of Peptide Depletion [%]

CV of Peptide Depletion [%]

Positive Control

1861.8433

0.2304

55.78

54.93

0.78

1.42

1926.2322

0.2384

54.25

1904.2300

0.2357

54.77

Test Item

3385.4136

0.4205

19.59

19.69

0.81

4.11

3345.2500

0.4155

20.55

3413.0500

0.4240

18.93

Prediction Model 1

Cysteine 1:10/ Lysine 1:50 Prediction Model1

Mean Cysteine andLysine PPD

ReactivityClass

DPRA Prediction²

0.00% PPD 6.38%

No or Minimal Reactivity

Negative

6.38% < PPD 22.62%

Low Reactivity

Positive

22.62% < PPD 42.47%

Moderate Reactivity

42.47% < PPD 100%

High Reactivity

1 The numbers refer to statistically generated threshold values and are not related to the precision of the measurement.

2 DPRA predictions should be considered in the framework of an IATA.

Prediction Model 2

Cysteine 1:10 prediction model1

Cysteine PPD

ReactivityClass

DPRA Predictio

0.00% PPD 13.89%

No or Minimal Reactivity

Negative

13.89% < PPD 23.09%

Low Reactivity

Positive

23.09% < PPD98.24%

Moderate Reactivity

98.24% < PPD 100%

High Reactivity

1 The numbers refer to statistically generated threshold values and are not related to the precision of the measurement.

2 DPRA predictions should be considered in the framework of an IATA.

 

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
Conclusions:
In this study under the given conditions the test item showed low reactivity towards the peptides. The test item is considered to potentially have a sensitizing property but the data generated with this test should be considered in the context of integrated approached such as IATA, combining the result with other complementary information, e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP.
Executive summary:

In the present study N-acetylhexanelactam was dissolved in acetonitrile. Based on a molecular weight of 155.2 g/mol a 100 mM stock solution was prepared. The test item solutions were tested by incubating the samples with the peptides containing either cysteine or lysine for 24 ± 2 h at 25 ± 2.5 °C. Subsequently samples were analysed by HPLC.

For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the cysteine peptide solution.After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples of the cysteine peptide run were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for the test item samples. A slight precipitation was observed for standard 1 and the positive control samples. Since the acceptance criteria for the linearity of the standard curve as well as for the depletion range of the positive control were fulfilled, the observed precipitations were regarded as insignificant.

For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the lysine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples of the lysine peptide run were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for any sample.

No significant co-elution of test item with the peptide peaks was observed. Sensitizing potential of the test item was predicted from the mean peptide depletion of both peptides by comparing the peptide concentration of the test item samples to the corresponding reference control C (RC C).

In the lysine run a shift of the lysine peptide peak from 7.7 min (STD 1) to 7.0 (REF C2 ACN) up to 6.5 min (REF B6) was observed (see16.4.2). Since the reference control samples allow clear identification of the lysine peptide and since the test item did not elute between 6.5 and 7.7 min, that indeed the lysine peptide peak had shifted and that the peak area of that peak did only represent remaining lysine peptide. Furthermore, all validity criteria were fulfilled, and this shift was not considered having an influence on the quality or validity of the results.

The 100 mM stock solution of the test item showed low reactivity towards the synthetic peptides. The mean depletion of both peptides was >6.38% (11.75%). Based on the prediction model 1 the test item is considered to potentially have a sensitizing property.

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 64.92%.

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